ERK2 enters the nucleus by a carrier-independent mechanism

doi:10.1073/pnas.112495999

ERK2 enters the nucleus by a carrier-independent mechanism

^*Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9041; and ^§Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030

Edited by Günter Blobel, The Rockefeller University, New York, NY, and approved April 3, 2002 (received for review September 19, 2001)

Abstract

In stimulated cells, the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) concentrates in the nucleus. Evidence exists for CRM1-dependent, mitogen-activated protein kinase kinase-mediated nuclear export of ERK2, but its mechanism of nuclear entry is not understood. To determine requirements for nuclear transport, we tagged ERK2 with green fluorescent protein (GFP) and examined its nuclear uptake by using an in vitro import assay. GFP-ERK2 entered the nucleus in a saturable, time- and temperature-dependent manner. Entry of GFP-ERK2, like that of ERK2, required neither energy nor transport factors and was visible within minutes. The nuclear uptake of GFP-ERK2 was inhibited by wheat germ agglutinin, which blocks nuclear entry by binding to carbohydrate moieties on nuclear pore complex proteins. The nuclear uptake of GFP-ERK2 also was reduced by excess amounts of recombinant transport factors. These findings suggest that ERK2 competes with transport factors for binding to nucleoporins, which mediate the entry and exit of transport factors. In support of this hypothesis, we showed that ERK2 binds directly to a purified nucleoporin. Our data suggest that GFP-ERK2 enters the nucleus by a saturable, facilitated mechanism, distinct from a carrier- and energy-dependent import mechanism and involves a direct interaction with nuclear pore complex proteins.

mitogen-activated protein kinase‖import‖FXFG motif‖nucleoporins

Footnotes

↵ † Present address: Department of Cell Biology, Harvard Medical School, Boston, MA 02115.
↵ ‡ Present address: Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226.
↵ ¶ To whom reprint requests should be addressed. E-mail: mcobb{at}mednet.swmed.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

MAP,

mitogen-activated protein;

MEK,

mitogen-activated protein kinase kinase;

ERK,

extracellular signal-regulated kinase;

JNK2,

Jun N-terminal kinase 2;

GFP,

green fluorescent protein;

GST,

glutathione S-transferase;

NPC,

nuclear pore complex;

NLS,

nuclear localization sequence;

TRITC,

tetramethylrhodamine B isothiocyanate;

WGA,

wheat germ agglutinin